The invention relates to the area of cancer diagnostics. More particularly, the invention relates to detection of the loss and or alteration of wild-type huBUB1 genes in tumor tissues.
Genes and proteins involved in cell cycle regulation and apoptosis have been found to be important in the development of cancers. There is a continuing need in the art for identification of components of cells which control the cell cycle and apoptosis.
The object of this invention is to provide tools and methods for diagnosing, prognosing, and treating neoplasia. This and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention provides an isolated and purified huBUB1 protein comprising an amino acid sequence which is at least 85% identical to the amino acid sequence shown in SEQ ID NO:2.
Another embodiment of the invention provides an isolated and purified huBUB1 polypeptide which comprises at least 6 contiguous amino acids selected from an amino acid sequence which is at least 85% identical to the amino acid sequence shown in SEQ ID NO:2.
Yet another embodiment of the invention provides a fusion protein comprising a first protein segment and a second protein segment fused together with a peptide bond. The first protein segment comprises at least 6 contiguous amino acids of a huBUB1 protein having an amino acid sequence which is at least 85% identical to the amino acid sequence shown in SEQ ID NO:2.
Still another embodiment of the invention provides a preparation of antibodies which specifically binds to a huBUB1 protein.
Even another embodiment of the invention provides an isolated and purified subgenomic polynucleotide comprising at least 10 contiguous nucleotides selected from a nucleotide sequence which is at least 85% identical to the nucleotide sequence shown in SEQ ID NO:1.
A further embodiment of the invention provides a DNA expression construct comprising an isolated and purified subgenomic polynucleotide. The isolated and purified subgenomic polynucleotide comprises at least 10 contiguous nucleotides selected from a nucleotide sequence which is at least 85% identical to the nucleotide sequence shown in SEQ ID NO:1.
Another embodiment of the invention provides a host cell. The host cell comprises an isolated and purified subgenomic polynucleotide. The isolated and purified subgenomic polynucleotide comprises at least 10 contiguous nucleotides selected from a nucleotide sequence which is at least 85% identical to the nucleotide sequence shown in SEQ ID NO: 1.
Still another embodiment of the invention provides a method of diagnosing a neoplastic tissue of a human. A tissue suspected of being neoplastic is isolated from a human. Loss of a wild-type huBUB1 gene or an expression product of a wild-type huBUB1 gene from the tissue is detected. The loss of the wild-type huBUB1 gene or its expression product indicates neoplasia of the tissue.
Yet another embodiment of the invention provides a method of supplying wild-type huBUB1 gene function to a cell which has lost said gene function by virtue of mutation in a huBUB1 gene. All or a portion of a wild-type huBUB1 gene is introduced into a cell which has lost said gene function. The portion of the wild-type huBUB1 gene is required for non-neoplastic growth of the cell. The all or a portion of the wild-type huBUB1 gene is expressed in the cell.
Even another embodiment of the invention provides a pair of single-stranded DNA primers. The pair of single-stranded DNA primers allows synthesis of all or part of a huBUB1 gene coding sequence.
Another embodiment of the invention provides a nucleic acid probe complementary to a wild-type huBUB1 gene as shown in SEQ ID NO:1.
Yet another embodiment of the invention provides a nucleic acid probe complementary to a mutant huBUB1 gene.
Still another embodiment of the invention provides a method of detecting the presence of a neoplastic tissue in a human. A body sample is isolated from a human. A mutant huBUB1 gene or expression product is detected in the body sample. Detection of a mutant huBUB1 gene or expression product indicates the presence of a neoplastic tissue in the human.
Another embodiment of the invention provides a method of detecting genetic predisposition to cancer in a human. A human sample selected from the group consisting of blood and fetal tissue is isolated. DNA is extracted from the sample. Loss of a wild-type huBUB1 gene from the DNA is detected. Detection of the loss of a wild-type huBUB1 gene indicates a genetic predisposition to cancer in the human.
A further embodiment of the invention provides a method for identifying test compounds which interfere with huBUB3-huBUB1 binding, said compounds being candidate therapeutic agents. A first protein, a second protein, and a test compound are contacted. The first protein comprises at least a portion of huBUB3 which binds to huBUB1 and the second protein comprises at least a portion of huBUB1 which binds to huBUB3 or the first protein comprises at least a portion of huBUB1 which binds to huBUB3 and the second protein comprises at least a portion of huBUB3 which binds to huBUB1. The quantity of the first protein which is bound to, is displaced from, or is prevented from binding to, the second protein or the quantity of the second protein which is bound to, displaced from, or is prevented from binding to the first protein is determined. A compound which diminishes the quantity of the first protein bound to the second protein or the second protein bound to the first protein, or which displaces the first protein bound to the second protein or the second protein bound to the first protein, or which prevents the first and second proteins from binding, is identified as a candidate therapeutic agent.
Another embodiment of the invention provides a method of identifying compounds which interfere with huBUB3-huBUB1 binding. A cell is contacted with a compound to be tested for its capacity to inhibit huBUB1-huBUB3 binding. The cell comprises a first fusion protein comprising a sequence-specific DNA-binding domain, a second fusion protein comprising a transcriptional activation domain, and a DNA construct comprising a reporter gene downstream from a DNA element which is recognized by the sequence-specific DNA binding-domain. The first fusion protein further comprises at least a portion of a huBUB3 protein which binds to a huBUB1 protein and the second fusion protein further comprises at least a portion of a huBUB1 protein which binds to a huBUB3 protein, or the first fusion protein further comprises at least a portion of a huBUB1 protein which binds to a huBUB3 protein and the second fusion protein further comprises at least a portion of a huBUB3 protein which binds to a huBUB1 protein. The amount of expression of the reporter gene in the presence of the compound is determined. A compound which decreases the amount of expression of the reporter gene is identified as a candidate therapeutic agent.
Still another embodiment of the invention provides a method of identifying test compounds which decrease the kinase activity of huBUB1. A huBUB1 protein is contacted with a test compound. The kinase activity of the huBUB1 protein is determined. A compound which decreases kinase activity of the huBUB1 protein is identified as a candidate therapeutic agent.
Even another embodiment of the invention provides a method of increasing the sensitivity of a tumor to an anti-tumor agent. The tumor is contacted with a compound which inhibits huBUB1 kinase activity or which inhibits huBUB1-huBUB3 binding. The sensitivity of the tumor to an anti-tumor agent is increased.
Another embodiment of the invention provides a method of expressing a huBUB1 subgenomic polynucleotide in a cell. The huBUB1 subgenomic polynucleotide is delivered to the cell. The huBUB1 subgenomic polynucleotide is expressed.
The present invention thus provides the art with the sequence of the human huBUB1 gene and protein. This information allows highly specific assays to be done to assess the neoplastic status of a particular tumor tissue.